Plants are natural sources of several bioactive substances, which have been found in extracts, secondary metabolites, and essential oils. Several biological activities have been attributed to essential oils as antiviral, insecticidal, antiparasitic, antioxidant, and antimicrobial. The indiscriminate use of antibiotics has increased the development of resistance mechanisms of microorganisms. Thus, search for efficient natural compounds with antimicrobial activity and low toxicity has increased, so essential oils have been a promising alternative for combating microbial infections. This study was carried out to investigate the seasonality effects on the infrared absorbance spectra, antibacterial activity, and antibiotic potentiating activity of essential oils from Vitex gardneriana leaves. Essential oils were extracted from V. gardneriana Schauer leaves the seasonal period from January to December 2016 and characterized by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The antibacterial effect of these oils and antibiotic potentiating activity, both determined by the minimum inhibitory concentration, were assessed using microtiter plates. For the first time, we present the use of infrared absorbance spectra of these essential oils and show the influence of seasonality on them. Synergistic effects were observed for the essential oils associated with the antibiotics tested (gentamicin, ampicillin, and ofloxacin). The main influence of seasonality on the infrared absorbance spectra of the essential oils of the V. gardneriana occurred in the June month (last month of the rainy season). In regard to antibacterial activity test, the essential oils of the V. gardneriana leaves did not show a direct effect on the strains tested. However, the essential oils when associated with the antibiotics showed variations in the minimum inhibitory concentration with the months of the seasonal period, indicating synergistic effects against Escherichia coli and Staphylococcus aureus bacterial resistance.