Antimicrobial peptides (AMPs) are potent compounds for treating bacterial infection and cancer, drawing ever-increasing interest. However, the function and mechanism of most AMPs remain to be explored. In this research, we focused on investigating the antibacterial and anticancer activities of four AMPs (Dhvar4, Lasioglossin-III, Macropin 1, and Temporin La) and the possible corresponding mechanisms. All four AMPs are cationic α-helical with moderate hydrophobicity and high helicity. They have broad-spectrum antibacterial capacities, among which the antibacterial activities of Dhvar4 and Temporin La are not as effective as Lasioglossin-III and Macropin 1. Macropin 1 exhibited the highest antibacterial effect with a pretty low minimal inhibitory concentration (MIC) of 2-8 μM. Meanwhile, Lasioglossin-III exhibited the strongest anticancer activities, displaying the IC50 of 26.36 μM for A549 and 7.75 μM for HepG2. Although Dhvar4 possessed the highest positive charge and entered the bacterial and animal cells in large amounts, it displayed the lowest bactericidal and anticancer activities which might be ascribed to its lowest hydrophobicity and thus the weakest cell membrane damage capability. It seems that the positive charge and cell internalization play a supporting rather than a determined role in antibacterial and anticancer activities of AMPs. All the four AMPs damaged the bacterial cell membrane with Macropin 1 damaging the cell membrane of Escherichia coli the most and Lasioglossin-III destroying the cell membrane of Staphylococcus aureus the worst. In addition, the animal cellular internalization of the four peptides was temperature-dependent and mainly mediated by caveolae-mediated endocytosis, and they were distributed in lysosomes once inside the cells. These findings expand our knowledge on the function and mechanism of AMPs, laying the fundamental theoretical basis for designing and engineering AMPs for infection and cancer treatment.