Background: Flavonoids from medicinal plants have been reported to be a possible alternative to synthetic drugs due to their antimicrobial activities. In the present study, flavonoids extracts from the leaves of Psidium guajava, Vernonia amygdalina, and Azadiracta indica were evaluated for antibacterial and anti-fungi activities against some microbial isolates from spoiled tomato fruit. Methodology: Food spoilage microorganisms were isolated from spoiled tomato fruit. Flavonoids were isolated from the leaves of P. guajava, V. amygdalina, and A. indica, and were evaluated for their inhibitory activities against the isolated microorganisms using standard protocols. Results: The flavonoids extract demonstrated dose-dependent antimicrobial activities. Flavonoids extracts from V. amygdalina exhibited an inhibitory effect on C. tropicalis, K. pneumonaie, M. roseus, and P. aeruginosa with maximum inhibition diameters (MID) of 17.00±0.05, 25.00±0.45, 15.00±0.05, and 16.00±0.34 mm while the flavonoids from P. guajava exhibited inhibitory effects on the same organism with MID of 15.00±0.05, 14.00±0.10, 16.00±0.55, 15.00±0.43 and 14.00±0.05 mm respectively, while the flavonoids extract from A. indica exhibited the least activity on the bacteria and fungi isolates. Results of the MICs and MBC revealed that the gram-positive bacteria tested (M. roseus & S. faecalis) were more susceptible to inhibition by the flavonoids extract of the plants. The combination of total flavonoids from leaves of P. guajava and V. amygdalina exhibited higher and synergetic antimicrobial activities against all the tested organisms when compared with individual flavonoids extracts. This combination could serve as a potential source of novel antimicrobial agents for food industry.