Three novel actinomycins, actimomycin S (1), neo-actinomycins C and D (2 and 3), and one new benzo[d]oxazole alkaloid (4) were isolated from the Streptomyces sp. strain S22, along with three known congeners F9 (5), X2 (6) and X0β (7) and 2-acetylamino-3-hydroxyl-4-methyl-benzoic acid methyl ester (8). The structures of the new products were elucidated by spectroscopic methods, and the absolute configuration of amino acid residues was determined by Marfey's analysis. Actinomycin S contains an aspartic acid (Asp) residue in the β-peptidolactone ring. This is the first report of an Asp residue within an actinomycin-type natural product. Notably, neo-actinomycins C and D feature a rare tetracyclic 5H-oxazolo[4,5-b]phenoxazine chromophore. Among these, neo-actinomycin D, with an unprecedented molecular formula, represents the highest molecular weight member in the actinomycin family. Actinomycins 1-3 exhibited antimicrobial activity against multiple resistant "ESKAPE" pathogens with MIC values ranging from 1.25 to 80.0 μg mL-1. In addition, 1-3 showed potent cytotoxic activities against the HepG2 liver carcinoma cell line with IC50 values of 0.10, 0.32, and 0.024 μM, respectively. Furthermore, 1 inhibited cell proliferation by inducing G0-G1 phase arrest in the cell cycle.