The yield of Ananas comosus (pineapple) is susceptible to microbial infection and the usage of chemical pesticides to control it has been often associated with negative impacts to the environment and human health. As pineapple leaves is one of the lavish organic materials with antimicrobial properties due to its total phenolic content (TPC), its potential as a microbial inhibitor is explored in this study. The objective of this research is to study the factors that affect microbe growth inhibition using pineapple leaves juice (PLJ). The factors evaluated were; reaction time between mixtures of PLJ and microbe (0.5 – 5 hours), concentration of TPC in PLJ (0.2563 – 0.5127 mg GAE/ml), reaction temperature (26 – 37°C), and ratio of microbe to PLJ (M/PLJ) (1:1 and 1:3). A two level factorial design was adopted to assess the effect of the above mentioned factors on the microbial inhibition by PLJ. The results show that the most contributing factor of 1.55 % was reaction temperature, meanwhile the highest contribution factor for interaction effect was between concentration of TPC in PLJ and ratio of microbe to PLJ at 5.17%. The best condition for microbe growth inhibition of 20.90% was found to be at reaction time of 0.5 hour, TPC in PLJ of 0.5127 mg GAE/ml, reaction temperature of 37°C, and M/PLJ at 1:1. This study demonstrates that pineapple leaves could be exploited as valuable sources of natural products that could be used as microbial growth inhibitor and thus become one of the cheap and green alternatives for more expensive chemical pesticides.