The Russian had never previously enjoyed a World Cup victory but he made sure to change all that in the ninth competition of the season.
Having progressed comfortably through qualifying of the 1.6km sprint, Ustiugov carried his form over into the final, crossing the line in a time of 2:54.59 – 0.18 seconds ahead of Federico Pellegrino of Italy.
Alexey Petukhov then made sure Russia had two representatives on the medal rostrum when he came home in third place.
The result moves Ustiugov up to second in the sprint standings, topped by Pellegrino, and tenth in the overall World Cup standings behind leader Martin Johnsrud Sundby.
Meanwhile in the women's sprint event, USA's Kikkan Randall took her first win of the season and first at Nove Mesto when she crossed the line in a time of 2:44.04.
Laurien Van der Graaf secured the best result for a Swiss woman since Evi Kratzer won in Calgary in 1987 when she came home in second while Ingvild Flugstad Oestberg finished third for Norway.
It leaves the Norwegian second in the sprint standings behind Germany's Denise Herrmann – fifth in the Czech Republic – with Norway's Therese Johaug continuing to top the overall standings.