During the course of studies on bioactive constituents of marine-derived microorganisms, a crude extract from a small-scale culture (100 ml) of the marine-mudflat-derived fungus Fusarium oxysporum was found to exhibit in vitro antimicrobial activity against methicillin-resistant and multidrug-resistant Staphylococcus aureus (MRSA and MDRSA) in a primary screen. A large-scale culture (10 l) was used to purify antibacterial metabolites from the broth extract, leading to the isolation of a new antibacterial alkaloid, oxysporizoline (1), a tris-anhydrotetramer of anthranilic acid (α-aminobenzoic acid), along with the known alkaloid 1H-indol-3-butanamide (2) and two polyketides, chlamydosporol (3) and butenolide (4-acetamido-4-hydroxy-2-butenoic acid γ-lactone) (4). The structural elucidation of these compounds is reported herein. The fungal strain F. oxysporum, isolated from marine mudflat in Suncheon Bay, Korea, was identified via 18S rRNA analysis (98% identity) and deposited at Pukyong National University (code MSA392). Purification involved Si gel flash chromatography, medium-pressure liquid chromatography (ODS column), and HPLC. Oxysporizoline (1), a colorless amorphous solid, was determined to have the molecular formula C28H22N4O2 by HR-EI-MS and 13C NMR. Its structure, N-(7,11b-dihydro-13H-6,12-o-benzeno-6H-quinazolino[3,4-a]quinazolin-13-yl)anthranilic acid, was elucidated using IR, 1H/13C NMR, and 2D-NMR (DEPT, COSY, HMQC, HMBC, NOESY), with relative configurations at C-6′, C-11b′, and C-13′ assigned as 6′S*, 11b′R*, and 13′S* via NOESY and molecular modeling. Biological activity assays showed that compounds 1 and 3 had weak antibacterial activity against MRSA and MDRSA with MICs of 6.25 μg ml−1 and 31.5 μg ml−1, respectively. Compounds 1 and 4 exhibited moderate DPPH radical scavenging activity with IC50 values of 10 μM and 12 μM, respectively, which were more active than the positive control ascorbic acid (IC50 20 μM). Given the biological activities of metal complexes derived from similar polycyclic quinazoline alkaloids (e.g., nuclease, superoxide dismutase, and antineoplastic activities), oxysporizoline (1) warrants further study for potential chemotherapeutic development against cancer and diseases related to nucleases and superoxide dismutase.