This study investigated the potential role of lactobacilli in mitigating the negative effects of heat stress on the functional activity of chicken macrophages. Macrophage-like MQ-NCSU cells were incubated at 40 and 44 °C in the presence or absence of a single or a mixture of different poultry-derived Lactobacillus spp., including Lactobacillus animalis, L. acidophilus, L. reuteri, and L. crispatus. Macrophage activation was evaluated by measuring nitric oxide (NO) production, phagocytic activity, and the transcription levels of cytokines, chemokine, and Toll-like receptors (TLRs). Macrophages exposed to heat stress exhibited increased production of NO, diminished expression of interleukin (IL)-1β and IL-12p40, and elevated expression of TLR2 and TLR4, whereas no significant alterations in the phagocytic activity of macrophages were observed. Conversely, treatment of macrophages with probiotic lactobacilli counteracted the effects associated with heat stress. This was evidenced by a notable enhancement in macrophage phagocytic activity, NO production and expression of IL-1β, IL-12p40, IL-18, and chemokine CXCL8, coupled with a reduction in TLR2 and TLR4 expression. These findings suggest that probiotic lactobacilli could be given to chickens to mitigate the negative effects of heat stress on their innate immune system. However, further studies are required to validate the observed effects in an in vivo model.