Modification of polyurethane foams composite by vanillin and thiourea is a search for a new antimicrobial active composite. TPU/Vanillin, a polyurethane foam composite loaded with vanillin and thiourea, was produced and tested for antimicrobial activity. TPU/Vanillin composite was synthesized by combining the surface qualities of polyurethane foams with the biological functions of thiourea and vanillin. TPU/Vanillin composite was identified by IR, UV-Vis, bandgap energy, magnetic susceptibility, chemical stability and pHPZC. The antibacterial and antifungal properties of TPU/Vanillin composite were investigated. TPU/Vanillin composite was tested in vitro against Gram-negative bacteria, Escherichia coli, Gram-positive bacteria, Bacillus cereus, and fungus, Aspergillus niger. Agar well diffusion, minimum inhibition concentration (MIC) and minimum microbicidal concentration (MMC) tested were used to determine the sensitivity of the tested microbes to the prepared composite. Zones of inhibition of TPU/Vanillin were 16 ± 0.12, 15 ± 0.19 and 25 ± 0.20 mm against E. coli, B. cereus, and A. niger, respectively. Successfully inhibiting the growth of microbial strains with MIC values of 130, 140 and 40 µg/ml against E. coli, B. cereus, and A. niger, respectively, proved that TPU/Vanillin composite could be further improved for potential applications such as food packaging and coating.