Pyrolysis of cajuput (Melaleuca leucadendron) twigs and rice (Oryza sativa) husks to produce liquid smoke and antibacterial activities of the liquid smoke fractions were investigated. The liquid smoke was produced by pyrolysis at 500 °C for 8 h and contained fine chemicals, such as acetic acid, carbonyl, cyclic ketones, and phenolic compounds with pH 2.1-2.9. The liquid smoke was separated by vacuum evaporation under vacuum conditions at low temperatures (40 °C, 50 °C, and 60 °C) to recover three fractions. The composition of each fraction influenced its antibacterial activities. Antibacterial activities of the liquid smoke fractions were tested against Gram-positive bacteria (Listeria monocytogenes, Bacillus subtilis, and Staphylococcus aureus) and Gram-negative bacteria (Salmonella typhimurium, Pseudomonas aeruginosa, and Escherichia coli). Whole fractions of the liquid smoke inhibited the six pathogenic bacteria, with the inhibition zone larger or smaller than the positive control. Among the liquid smoke fractions, the liquid recovered at 60 °C for the cajuput twigs and rice husks demonstrated a stronger inhibitory effect on bacterial growth than the other fractions.