The research aimed to develop composite materials based on waste cooking oil containing thymol or salicylic acid and assess their antimicrobial properties against selected Gram-positive and Gram-negative bacteria strains. The study also investigated whether the use of a binder, such as catalyzed waste cooking oil, and the performing of annealing process would affect the antimicrobial effectiveness of the natural additive. The composite materials were characterized using FT–IR spectroscopy, thermal analysis, and scanning electron microscopy. Mechanical properties were also evaluated, along with abrasion resistance and soakability. Thymol-modified materials showed higher activity against E. coli strain; while, salicylic acid-modified materials were more effective against P. aeruginosa. The antibacterial activity against Gram-positive bacteria was generally lower than against Gram-negative bacteria. Flow cytometry and confocal microscopy were used to visualize and quantify bacterial death induced by the composite materials. The materials showed interactions with bacterial cells, leading to cell damage and inhibition of bacterial division. The most effective biocidal composite against the suspension of P. aeruginosa bacteria was 22 T (99% inactivation), containing 4% thymol. Against E. coli, composite 41 T containing 1% thymol caused a significant decrease in the viability of these bacteria by up to 45%. Similarly, on S. aureus, composites with the addition of thymol also exhibited strong effects, reaching up to 70% reduction, as observed in 43 T with 7% thymol. Composites containing salicylic acid also demonstrated biocidal properties, resulting in a 52% reduction in E. coli (33SA containing 7% salicylic acid); 99% reduction in P. aeruginosa (15SA containing 1% salicylic acid); 20% reduction in S. aureus (41SA containing 1% salicylic acid); and approximately 25% reduction in S. epidermidis (43SA with 7% salicylic acid). Furthermore, the composite materials demonstrated low cytotoxicity against human keratinocytes, indicating their potential safe use when contacted with human skin.
