A second half penalty by Albie Hall settled a tight encounter at Butlin Road in favour of Newport Pagnell Town.
Hall's solitary goal extended Valley's run of home games without a win to three, whilst simultaneously denting their league title aspirations in the process.
Tom Fielding was preferred to Ryan Seal in an advanced midfield role in the only change to the starting line-up from the 4-4 draw at Oadby the previous weekend.
Kicking against the wind in the first half, Rugby found themselves under some early pressure, and the first chance of the game saw Nathan Tshikuna put one well wide for the visitors.
Ben Shepherd's low strike then drew a decent one-handed save from Matt Hill, as Newport continued to edge the opening exchanges.
Jordan Wilson nearly broke through the Pagnell back-line in a rare attacking threat from the home side, but was dispossessed by a good covering tackle, before Hill was back in the action smothering Kieran Barnes's effort at the second attempt after Hall's initial effort had been blocked.
Town did start to establish more of a foothold on the game though, and Fielding smashed the ball into net after Caine Elliott had floated in a free-kick, but the official's flag had been raised in the lead up to this.
Luke English nodded on another Elliott set-piece but the ball drifted just beyond Liam Francis, shortly before Valley very nearly breached the well-organised away defence, when Wilson's header to a Fielding whipped-in cross bounced back off the inside of the post.
David Kolodynski's hooked shot from a tight angle was comfortably collected by Newport keeper Martin Conway, before the visitors ended the first half in the ascendance with Jake Watkinson, Alfie Powell and Barnes all missing out with a series of chances.
The first opportunity of the new period saw an English cross fly just over the head of Kolodynski at the far post, but the game's decisive moment came when Keenah Rosser lost control of the ball to allow Watkinson a run into the box - which Rosser ended illegally with a clumsy challenge from behind.
Hall duly stepped up to send Hill the wrong way with his low spot-kick into the bottom corner.
A Wilson strike was deflected across the face of the Newport goal, with Loyiso Recci's header from the resulting corner cleared away from the goalmouth, as Valley looked for an instant response.
Kolodynski's low swerving effort from 25 yards was also pushed away by Conway's body, but there was no-one on-hand to capitalise on the rebound.
Generally Pagnell coped ably with Town's efforts thereafter, and it was the visitors that looked the more likely candidates to score again.
Hall had two shots blocked and Shepherd's low strike was saved by Hill in the same passage of play, with Valley struggling to penetrate at the other end - as all too often their deliveries into the box either drifted harmlessly behind or were too close to Conway and easily caught.
Mike Taylor did mark his first appearance in a Rugby shirt from the bench with a couple of crosses which carried a little more threat, but ultimately Newport saw out the game to register their first victory against Valley at the sixth attempt in the UCL.