The seafood industry is often left out of the food waste discussion, but this sector is no exception, as it generates large amounts of various by-products. This study aimed to explore the potential of the microwave-assisted extraction (MAE) technique to obtain high-quality oil from fish by-products. The independent variables, which were time (1-30min), microwave power (50-1000 W), and solid/liquid ratio (70-120g/L) were combined in a 20-run experimental design coupled with the response surface methodology (RSM) for process optimization. The obtained oil yield values were fitted to a quadratic equation to build the theoretical models, which were statistically validated based on statistical criteria and used to predict the optimal MAE condition. The oil yields were significantly affected by the three independent variables through linear, quadratic, and/or interactive effects. Compared to a conventional Soxhlet extraction (SE), the optimal MAE conditions allowed between 60 and 100% of oil to be recovered in less than 19 min and with less solvent consumption. The fatty acid profiles of the oils obtained through SE and optimized MAE were characterized by gas chromatography with flame ionizing detection (GC-FID) after a derivatization process. These oils were constituted mainly of health, beneficial unsaturated fatty acids, such as oleic, docosahexaenoic (DHA), linoleic, and eicosapentaenoic (EPA) acids, which were not affected (p > 0.05) by the extraction methods. Interestingly, the oils obtained through MAE showed the best microbial growth inhibition results may have been due to thermolabile compounds, preserved via this unconventional non-thermal method. The oils also exhibited anti-inflammatory effects via nitric oxide production inhibition and cytotoxic potential especially, against breast and gastric adenocarcinoma cells. However, the threshold of toxicity should be further investigated. Overall, this work emerges as a future-oriented approach to upcycling fish by-products into high-quality oils that can be used in the formulation of pet food and other products. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.