Low molecular weight protease inhibitors are useful as medicines or reagents for research. During screening for thiol protease-specific inhibitors from culture filtrates of microorganisms, the authors previously isolated estatins A and B, and further screened for inhibitors against cathepsin B, discovering new inhibitors AM4299 A and B from the culture filtrate of Chromelosporium fulvum M4299 (isolated from a soil sample in Iriomote island, Okinawa Prefecture, Japan). This paper reports the isolations, physicochemical properties and inhibitory activities of AM4299 A and B. AM4299 A and B were isolated as white powder via column chromatography (carbon, ion-exchange resins, silica gel, etc.). They were stable at pH 2.0, 7.0, 9.0 at 60°C for 30 minutes, soluble in water, acetic acid, DMSO and pyridine, insoluble in most organic solvents. Positive Feigl and thiosulfate tests indicated 1,2 dicarboxylic acid and epoxide groups; AM4299 B showed positive ninhydrin reaction (amino group). IR spectra suggested amide, carbonyl, epoxide and amine groups. Molecular formulas were C15H26O6N2 (AM4299 A) and C16H27O7N3 (AM4299 B) via elemental analysis and FAB-MS. Hydrolysis revealed leucine in AM4299 A, and leucine plus lysine in AM4299 B. NMR (1H, 13C, COSY, HMBC) data led to proposed structures with amino acids and epoxysuccinate moieties, similar to E-64 and estatins but lacking agmatine moieties. AM4299 A and B had no antimicrobial activity (≤100μg/ml) and low toxicity (200 mg/kg iv to mice caused no death). They strongly inhibited cathepsin B, cathepsin L and papain, but not α-chymotrypsin or trypsin.