The continuous increase of bacterial pathogen resistance to conventional antibiotics has challenged the research community to develop new antimicrobial strategies. Antimicrobial peptides (AMP) are a promising alternative to combat multidrug-resistant strains compared to conventional antibiotics because of their biocompatibility. In the present study, the Flo peptide, an AMP from the Moringa oleifera tree, was expressed in the chloroplast of the microalgae Nannochloropsis oculata and Scenedesmus acutus. The transgene insertion was verified by PCR amplification, and the homoplasmy was corroborated in spectinomycin-resistant lines. The identification and quantification of the peptide were performed using ELISA. The antimicrobial activity was studied against the Gram-negative Escherichia coli (ATCC 25,922) and Klebsiella pneumoniae (ATCC 700,603). The inflammatory response of the total soluble proteins of transplastomic N. oculata was assessed by measuring secretion of the cytokines IL-6, IL-10, and alpha-tumor necrosis (TNF-α), and cytotoxicity was assessed. These results provide a potential strategy to produce the Flo peptide in microalgae with antibacterial activities.