A proper regulation of macrophage polarization is essential for the organism’s health and pathogen control. Differentiation control is known to occur at the transcriptional as well as the posttranscriptional levels. The mechanisms involved, however, have not yet been fully elucidated. In this study, we co-cultured macrophages with viable Gram-positive and Gram-negative bacteria to mimic macrophage differentiation to the M1-like type in an inflammatory milieu. We found that Gram-positive stimulation resulted in increased expressions of miR-7a-5p, miR-148a-3p, miR-155-5p, and miR-351-5p. Of note, these miRNAs were found to target inhibitory mediators of the Rac1-PI3K-Akt pathway and the MyD88-dependent pathway. In contrast, Gram-negative stimulation-induced downregulation of miR-9-5p, miR-27b-3p, miR-93-5p, and miR-106b-5p is known to target key members of the Rac1-PI3K-Akt pathway and the MyD88-dependent pathway. These results, taken together, point to a fine-tuning of macrophage polarization by TLR-induced changes in macrophage miRNA profiles. Here, the miRNA-mediated priming of M1 differentiation seems to differ in the Gram-positive and Gram-negative settings in terms of the mechanism and miRNAs involved.