The present study explains the growth pattern and polyhydroxyalkanoates (PHA) production capacity of Bacillus sp. CYR1 (DNA Data Bank of Japan accession number LC049103) uses various fatty acids as substrates. Various combinations of fatty acids were used as substrates for the growth of strain CYR1. Among them, strain CYR1 showed good growth with the combination of two fatty acids (acetic acid and butyric acid) and the combination of three kinds of fatty acids (acetic acid, propionic acid, and caproic acid). Apart from the growth pattern, PHA production was also evaluated using various fatty acids as substrates. PHA production was in coordination with the growth pattern. The amount of PHA and PHA production (% cell dry mass (CDM)) calculated based on the Soxhlet extraction method was 0.158 g/L and 41.8 (%CDM) for the combination of acetic acid and butyric acid, and 0.241 g/L and 40.16 (%CDM) for the combination of acetic acid, propionic acid, and caproic acid. The wet and dry weights were higher in the combination of the three types of combination, and the amount of PHA was higher in the combination. It is essential to reduce the cost of the substrate for PHA production by replacing expensive carbon sources with wastewater. The organic compounds in domestic wastewater and wastewater produced from various factories will contain mixed fatty acids. By studying different combinations of two or more fatty acids, we gained new insights into utilizing wastewater containing various fatty acids.