Citrus oils obtained from citrus by-products are widely used in many areas because of their bioactive nature. However, their use in products has been restricted due to phototoxicity and other health problems of some of the non-volatile compounds, especially bergapten. We studied the effect of supercritical carbon dioxide (SC-CO2) extraction conditions on the minimization of bergapten content for the oils obtained from a combination of citrus peels and seeds. The influence of combined citrus by-products on cytotoxicity, antimicrobial activity, and other quality characteristics was studied so that those by-products can be used effectively. A second-order polynomial model showed a suitable fitting of the experimental value for the bergapten content (R2 = 0.977, p < 0.05). Optimization (minimization of bergapten content) and validation were performed. The optimum conditions were 200.54 bar, 46.28°C and 34.98 g/min for pressure, temperature, and flow rate, respectively. The corresponding predicted value was 37.82 μg/g oil, which agreed well with the experimental value (38.36 ± 0.44 μg/g oil), affirming the adequacy and validity of the predicted model. The oil displayed higher antimicrobial activity and was less susceptible to fungi than bacteria. The cytotoxicity of the oil on HaCaT cells was low (84.59 ± 1.24% cell viability) for a concentration of 500 μg/mL. SC-CO2 extraction of a mixture of citrus by-products can yield oils with low bergapten content, low toxicity, and higher antimicrobial activity, which could have a variety of applications.