Pistorius finished second in 45.52 seconds in the final — his last qualifying race — but was again outside the 45.30 he needed to run once more at an international event to meet his national federation's qualifying criteria for next month's games.
Pistorius could still make history as the first amputee track athlete at the Olympics if he's selected for South Africa's 4x400 relay team.
He clocked an Olympic-qualifying time of 45.20 in Pretoria in March but had to do it again outside his home country before Saturday's cutoff to be picked for the individual 400.
The 25-year-old who runs on carbon fiber blades made it his mission to compete at an Olympics and Paralympics in the same year, and said he was still hopeful of being picked for the relay team.
Pistorius helped South Africa win silver in the 4x400 last year in an historic first appearance for an amputee runner at the world championships.
"My race today felt good and I'm pleased to have won the silver medal at the African Championships," Pistorius said from Porto Novo. "I am obviously disappointed that my time was just outside of the Olympic qualification time by two tenths of a second, I had felt very strong coming into this competition as my fitness and speed has been continually improving. I was in good shape to set the time.
"I had a great early start to the season, setting the Olympic qualification time and I am hoping that there is still the opportunity for me to be selected to run for South Africa in the 4x400m relay."
The relay team will be picked on form in July, according to Athletics South Africa, and Pistorius is likely to be included after his 45.20 at the start of the season. He has been South Africa's best-performing 400-meter runner this year despite him missing out on the crucial second Olympic time.
Pistorius was already selected in South Africa's Paralympic team to defend his 100, 200 and 400 T44 titles in the British capital.