A potential novel genus of microalgae was isolated from swine wastewater. According to a phylogenetic analysis of 18S rRNA and internal transcribed spacer (ITS) sequences, as well as the ITS2 secondary structure, this strain was designated as Chlorellaceae sp. P5. Acclimatization to various temperatures, pH conditions, and salinities demonstrated that this strain can grow at temperatures of 20 °C–40 °C, in an acidity range of pH 4–7, and with salinity as high as 3% NaCl. To optimize biomass production, response surface methodology was used which included variables associated with carbon and nitrogen sources. In this study, flask cultivation yielded 1.66 g l−1 d−1 of biomass under 15.19 g l−1 glucose and 8.26 mM ammonium bicarbonate. The fatty acid composition suggested that the lipid from Chlorellaceae sp. P5 can be typically used for biodiesel production, with a low degree of unsaturated fatty acids. Moreover, a high content of the first-limiting amino acid (lysine) and 9,12-Octadecadienoic acid (linoleic acid, C18:2) for nutritional supplements was also observed. With respect to swine wastewater treatment, the isolated strain showed an excellent removal rate of ammonia and phosphate over 97.3 mg l−1 of NH3-N and 20.3 mg of l−1 PO43− in the sterilized and none-sterilized wastewater. The biomass and lipid derived from Chlorellaceae sp. P5 can be attained at 0.49 g l−1 d−1 and 0.13 g l−1 d−1 by swine wastewater cultivation with 5% CO2 supply, respectively. According to the various characteristic analyses, Chlorellaceae sp. P5 has great potential for different industrial developments such as biodiesel and wastewater treatment.