Alternative energies have become relevant in global strategies to address climate change, and third-generation biodiesel derived from the generation of lipids from microalgae represents a viable option. This process can also be coupled with wastewater treatment to remove organic matter. To determine the effects of two catalyst levels (1 and 1.5% KOH) and two molar ratios of alcohol (methanol) with oil (1:6 and 1:9) on the conversion of lipids into FAMEs and the quality of the biodiesel produced, this work suggests a method for the ultrasonication-based extraction of lipids from C. vulgaris. It also employs an experimental 2² design and three replicates. It was found that with a molar ratio of 1:9 and a 1% catalyst, the highest yield of 98.48 ± 1.13% was achieved. The FAME profile was similar to the profiles obtained in cultures with bold basal medium or INETI. The quality of the biodiesel met ASTM standards, achieving refractive indices of 1.435–1.478. The flash point (FP) was 165 ± 18 °C, and the acid number was 0.31 ± 0.17 mg KOH/g. The viscosity ranged from 4.33 to 4.87 mm²/s. However, the rheological behavior was correlated with the Ostwald–de Waele model with pseudoplastic behavior.