Justicia pectoralis Jacq. (Acanthaceae) leaves currently found in the Brazilian north-east are widely used to treat diabetes, menstrual pains, asthma, and other disorders. This work aimed to identify the phytochemical characterization and biological activities of J. pectoralis leaf extracts. The plant material was ground and the crude extracts were obtained with water or acetone: water (7:3 v/v), yielding aqueous (JPA), and organic (JPO) extracts. Phytochemical characterization was performed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Cytotoxicity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay and trypan blue (TB) exclusion assay in peripheral blood mononuclear cells (PBMCs), BALB/c splenocytes, and neoplastic cells (TOLEDO, K562, DU-145, and PANC-1) at 1, 10, and 100 μg/mL. Antibacterial activity was evaluated using the microdilution test to obtain the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Cytokines, IFN-γ, and IL-17A from culture supernatants of BALB/c mice splenocytes were measured by sandwich ELISA. In the TLC analysis, both JPA and JPO extracts presented coumarin and flavonoids. In addition, HPLC was able to identify coumarin, apigenin, and ellagic acid in both extracts. JPO IC50 was 57.59 ± 1.03 μg/mL (MTT) and 69.44 ± 8.08 μg/mL (TB) in TOLEDO. MIC value of JPO against Acinetobacter baumannii and Klebsiella pneumoniae was 500 μg/mL. JPO (100 μg/mL) significantly inhibited IFN-γ levels (p=0.03). J. pectoralis is a potential candidate to be further investigated as an IFN-γ inhibitory agent and against Acinetobacter baumannii and Klebsiella pneumoniae.