This study aims to optimize the extraction conditions to obtain the highest yield, to characterize tomato peel extract (TPE) under optimized conditions, and also to determine the effect of ambient oxygen on the properties of TPE. Optimisation were performed at three temperatures (60 °C, 80 °C, 100 °C) and three periods (2, 4, 6 h) by the response surface methodology. The properties of the extract under atmospheric and oxygen-free conditions (AC, OFC) were analysed to determine whether the characteristics of both extracts changed depending on the presence of oxygen; moreover, the morphological, chemical, thermal, biochemical, and antimicrobial properties were analysed. The maximum yield was 31.3% at 100 °C/6 h. A quadratic model was used to create the best fit. Both TPE samples exhibited similar morphological structure, similar weight losses at three stages of TGA curve, similar band assignments in FTIR spectra. GC-MS analysis showed that both samples mainly consisted of cutin in abundance of 70.45% and 68.14% for AC and OFC, respectively. OFC had higher total phenolic content possibly depending on the absence of oxygen. AC and OFC extracts exhibited substantial antimicrobial activity against S. aureus, E. coli, C. albicans, and A. brasiliensis with a MIC value of 100 μg TPE/ mL.