This study compared the antimicrobial activity and total phenolic content of three Pinus plants (Pinus densiflora, P. thunbergii, P. rigida) for the first time. The antimicrobial activity of the water fraction of methanol extract of fresh leaves was stronger than that of fallen leaves at any concentrations. The water fraction of crude methanol extract from fresh leaves of P. thunbergii showed a higher growth inhibitory activity against gram-positive and gram-negative bacteria than that of P. densiflora and P. rigida. The results from the disc diffusion method followed by measurements of minimal inhibition concentration (MIC) indicate that Bacillus subtilis was the most sensitive microorganism with the lowest MIC value. The highest total phenolic content was found in fresh leaves of P. rigida and P. thunbergii. The assay showed that the fresh leaves of the three Pinus plants contained higher total phenolic content than fallen leaves of the three plants. The antimicrobial activity was related with the total phenolic content.