The aim of the present study was to assess the effect of sourdough related parameters on the growth and plantaricin activity of six Lactiplantibacillus plantarum strains against a mixture of 5 Listeria monocytogenes strains and to analyze the transcriptomic response of their pln genes. Parameters included 3 substrates (MRS broth, mMRS broth, WFE), 3 temperatures (20, 30, 37 °C), 2 initial pH values (5.0, 6.0), 2 NaCl concentrations (0.0, 1.8%) and 12 time points (ranging from 0 to 33 h). The transcriptomic response of the plantaricin genes to the aforementioned parameters was assessed after 21 h of growth. In general, plantaricin activity was strain dependent with that of Lp. plantarum strains LQC 2422, 2441, 2485 and 2516, harboring four pln genes, namely, pln423 (plαA), plαΒ, plαC and plαD, reaching 2560 AU/mL. However, strains LQC 2320 and 2520, in which 18 pln genes were detected, namely, plNC8a, plNC8b, plNC8c, plnL, plnR, plnJ, plnK, plnE, plnF, plnH, plnS, plnY, plNC8-IF, plNC8-HK, plnD, plnI, plnM and plnG, exhibited plantaricin activity barely reaching 160 AU/mL. Substrate, temperature, initial pH value and strains significantly affected plantaricin activity, while NaCl had only a marginal effect. Similarly, growth substrate and temperature had a more pronounced effect than initial pH value on gene transcription. A strong correlation between the transcription of the genes belonging to the same locus was observed; however, only a weak correlation, if any, was observed between plantaricin activity and the transcription of the genes assessed.