Just a week out from the US Open, Pennetta - the sixth seed at the WTA Tour event - was a 6-1 6-3 winner over Czech Koukalova after one hour two minutes in the opening round in New Haven on Sunday.
The 32-year-old - a quarter-finalist at the Australian Open in January - served up a total of five aces, with two of those coming in succession as she served out the match on the hard courts at the Connecticut Tennis Center.
Pennetta, who won 80 per cent of her first service points, will meet American Alison Riske for a spot in the third round.
Riske, 24, eliminated Australian world number 30 Casey Dellacqua 6-1 6-4 with four breaks of serve doing the damage.
After being broken in the opening game, Riske hit back with three breaks of her own to win the next games as she closed out the first set.
With scores level at 4-4 in the second, Riske broke again on her way to a resounding win.
Russian Ekaterina Makarova earned a second-round showdown with second Petra Kvitova following her routine win against Italy's Roberta Vinci.
Makarova - a semi-finalist in Montreal - won 6-3 6-3 and will now meet Kvitova, who was given a first-round bye.
Meanwhile, Italian Camila Giorgi rallied to beat American Coco Vandeweghe 6-3 6-1.