Powell, a former world record holder, powered across the finish line in 9.97 seconds before realising that Kemar Hyman of the Cayman Islands had been disqualified for jumping out of the blocs too quickly.
After wandering back to the start, Powell then kept his composure to win again, albeit in the slower time of 10.06 with Kim Collins of Saint Kitts and Nevis second in 10.08 and American Mike Rodgers third in 10.16.
Powell, who has struggled with a hamstring injury this season, had been looking to bounce back from the disappointment of a seventh place finish at the Jamaican trials in Kingston last week, where he clocked 10.22.
World record holder Usain Bolt, a regular at the Ostrava meeting in recent years, pulled out of the competition earlier this week, and Powell switched to the 100 metres from a relay he had initially committed too.
In the women's pole vault, world record holder Yelena Isinbayeva won with a leap of 4.78 metres, her best performance of the season but behind the world leading 4.90 of Cuba's Yarisley Silva.
There had been rumours Isinbayeva was considering retirement after she failed to win a third consecutive gold at the London Olympics last year, but she is planning to compete at the Moscow world championships in August.
The men's pole vault was won by France's Olympic champion Renaud Lavillenie, who improved the meeting record to 5.92 metres, just behind his world-leading 5.95 from Eugene earlier this month.
Olympic champion Tirunesh Dibaba of Ethiopia improved the season best in the 10,000 metres by clocking 30:26.67 while Czech Vitezslav Vesely won the javelin with a throw of 87.58 metres, just 0.02 off the best of the year by Finn Tero Pitkamaki.
- Sports & Recreation
- Kemar Hyman
- Yelena Isinbayeva