Thyme oil nanoemulsions (TONa and TONb), having 219 nm and 163 nm diameters, were successfully prepared by using sonication technique. Zeta potential (ZP) and polydispersity indexes (PDI) of TONa and TONb were defined to be 19.77, 0.24 and -24.80, 0.054, respectively. It was determined that zeta size values of TONs might have played a role on the inhibition of Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium. All bacteria tested by agar diffusion method were gram-negative bacteria that could be mostly found in fish fillets. Within 9 days cold storage period, TMAB growth in fish fillets treated with TONb was successfully reduced from 6.42 to 4.62 log CFU/g (change: 28%). In addition, as compared with control group samples, TPB growth of the same group was limited by 1.51 log CFU/g (from 7.93 to 6.42 log CFU/g). The results suggested that treatment of the fish fillets with the TONs would be a useful and practical nano application to delay microbial growth in fish fillets. Practical applications Thyme oil nanoemulsions (TONa and TONb) with a lower diameter than 219 nm have been successfully obtained. Antibacterial effects of TONa and TONb nanoemulsions have been revealed before treatment with fish fillets. The results revealed that TONs could be successfully used for the limitation of microbial spoilage of fish fillets stored at 4 degrees C for 9 days. In this respect, the nano application with rapid and cost-effective has been presented for the fishery processing industry.