Current research in the field of antimicrobials is focused on the development of novel antibiotics and antimicrobial agents to counteract the huge dilemma that the human population is mainly facing in regards to the rise of bacterial resistance and biofilm infections. Host Defense peptides (HDPs) are a promising group of molecules for antimicrobial development as they share unique characteristics suitable for antimicrobial activity including their broad spectrum of activity and potency against bacteria. AamAP1 is a novel HDP that was identified through molecular cloning from the venom of the North African scorpion Androctonus amoeruxi. In vitro antimicrobial assays revealed that the peptide displays moderate activity against different strains of Gram-positive and Gram-negative bacteria. Additionally, the peptide proved to be highly hemolytic and displaying significantly high toxicity against mammalian cells. In our study, a novel synthetic peptide analogue named A3 was designed from the naturally occurring scorpion venom host defense peptide. The design strategy depended on modifying the amino acid sequence of the parent peptide in order to increase its net positive charge, percentage helicity and optimize other physico-chemical parameters involved theoretically in HDPs activity. Accordingly, A3 was evaluated for its in vitro antimicrobial and anti-biofilm activity individually and in combination with four different types of conventional antibiotics against clinical isolates of multi-drug resistant (MDR) Gram-positive bacteria. A3 was also evaluated for its cytotoxicity against mammalian cells. A3 displayed potent and selective in vitro antimicrobial activities against a wide range of MDR Gram-positive bacteria. Our results also showed that combining A3 with conventional antibiotics displayed a synergistic mode of action which resulted in decreasing the MIC value for A3 peptide as low as 0.125 µM. These effective concentrations were associated with negligible toxicities on mammalian cells. In conclusion, A3 exhibits enhanced activity and selectivity when compared with the parent natural scorpion venom peptide. The combination of A3 with conventional antibiotics may be pursued as a potential novel treatment strategy against MDR and biofilm forming bacteria.