One of the most widely used medicinal plants in the world is Piper cubeba L., which is a member of the Piperaceae family. Identifying and isolating the bioactive components from P. cubeba L. fractions were the aims of this study. Additionally, Bacillus cereus ATCC33019, B. subtilis ATCC6633, B. pumilus ATCC14884, and B. megaterium ATCC14581 vegetative cells and spores were used to assess the isolated compounds' antibacterial and antispore properties. The separation of these compounds from the dichloromethane (DCM) and n-hexane fractions of the methanolic extract of P. cubeba L berries is described in this report for the first time. The n-hexane fraction yielded compound (1), which was separated, and the DCM soluble fraction yielded compound (2). These compounds were isolated using a variety of sequential chromatographic methods. The first compound is linoleic acid 1 while the second one is cubebin mixture 2. Anti-Bacillus activity of the obtained compounds were evaluated. The results demonstrated that exposure to compound (1) caused a wide inhibition zone with a diameter ranging from 9.31 to 9.61 mm in Bacillus sp. vegetative cells. The minimal bactericidal concentration (MBC) for Bacillus sp. was 250.0 g/mL, and the minimum inhibitory concentration (MIC) was 63.0 g/mL. After four hours of incubation, isolated chemical (1) at a concentration of 0.05% inactivated more than 3-Log10 (90.99%) of the Bacillus sp. spores, and at a concentration of 0.1%, all the spores were destroyed. Compound 1 demonstrated good antibacterial activity against vegetative cells and spores of Bacillus sp. However, compound 2 did not show any activity against Bacillus sp. Compound (1) may possess anti-Bacillus effects through its actions. To help increase food shelf life, it might be added to food products as well as preservatives.