The study was carried out to determine antimicrobial and haemolytic activities as well as to determine molecular weight breadth of Agelenopsis naevia venom components. Venom was extracted by micro-dissection. To measure the haemolytic activity, venom was incubated with 1% fresh human erythrocytes in 0.58 mg/ml, 2.54 mg/ml and 4.04 mg/ml for 1 hr, 2 hr, 4 hr and 6 hr while antimicrobial activity of venom was tested on Bacillus subtilis, Candida albicans, and Salmonella typhi. The venom protein molecular weights were determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoresis (2DE). The results show that haemolytic activity on human erythrocytes was dose-dependent and increased with time (42.40%–52.52%) with an effective concentration (EC50) of 2.07 mg/ml. The venom also showed significant (p < 0.05) activity against Bacillus subtilis (23.5 ± 0.50) compared to the positive and negative controls; however, there was no activity against Candida albicans or Salmonella typhi. SDS-PAGE revealed proteins/ peptides of molecular weights ranging between 6 kDa–97 kDa, while over 300 spots were detected on the 2D gel with molecular weight ranging from below 14 kDa–94 kDa and isoelectric point of 3–10 pI. This study concluded that A. naevia venom contains protein/peptides of various molecular weights with haemolytic activity on human erythrocytes and antimicrobial activity on Bacillus subtilis.