The antibiotic resistance is a natural phenomenon that can be considered harmful for humans when antibiotic resistant bacteria transfers resistance genes to the human pathogens at uncontrollable rate. One of the potential environments for spreading of antibiotic resistance is found to be in biogas reactors due to their mesophilic conditions that are optimal for the bacterial reproduction. The sudden changes in the environment of biogas reactors could negatively affect both biogas production rate but also microbial reproduction ability. This experimental research was done to determine to what extend does changes in pH with the increased volume dosing of acetic acid affect the persistence of antibiotic resistant enterobacteria in biogas reactors. Additionally, this experimental research is aiming to determine the difference in the enterobacterial persistence between year 2023 and year 2024 for the two biogas reactors (Selena and Taylor) fed with the chemically based substrate. The results show that the general persistence of antibiotic resistant enterobacteria in both reactors increased year 2024 compared to year 2023. According to the blank samples, the average persistence of enterobacteria increased in Selena by 500% and in Taylor by 250%. However, overall increase of enterobacteria that are antibiotic resistant cannot be determined where it can only be observed that the resistance has increased against majority of tested antibiotics. The obtained results also show a trend that in most tested groups quantity of antibiotic resistant enterobacteria is gradually increasing with the higher volume of acetic acid. The pH level decreased with the increased volume of acetic acid prior the incubation process with an average of 6,4 for Taylor biogas reactor and 6,7 for Selena biogas reactor. This indicates that the composition of fatty acid affected the environment so that it became more acidic. However, after the incubation process, the increased pH was detected in all groups compared to the results prior the incubation with the pH average of 8,1 for Taylor biogas reactor and 7,9 for Selena biogas reactor. This indicates that the acetic acid was consumed by the present bacteria and a decrease of acetic acid quantity resulted in an increase of pH. With that being said, the acetic acid can be considered as the enrichment substrate for the enterobacterial colonies adequate for efficient exchange of antibiotic resistant genes hence reproduction. However, the statistical analysis (nonparametric Kruskal-Wallis) results show that only between minority of tested groups the significant difference was detected.