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Pseudomonas putida MCCC 1A00316 was originally isolated from an Antarctic soil and has demonstrated potential nematicidal activity. Thus, it has promising applications for the biological control of Meloidogyne incognita. The larval mortality and egg-hatching inhibition rates of M. incognita will increase with the rising concentration of culture filtrates of P. putida MCCC 1A00316 and the duration of exposure. Thus, this study aimed to separate, purify, and identify nematicidal compounds from P. putida MCCC 1A00316 and to validate their anti-M. incognita activities. Compounds were purified through silica gel column chromatography and thin-layer chromatography combined with high-performance liquid chromatography (HPLC). Structural identification was conducted through liquid chromatography time-of-flight mass spectrometry, 1H nuclear magnetic resonance (NMR) spectroscopy, 13C-NMR, and Marfey’s method. The isolated compounds were identified as cyclo(l-Pro–l-Leu) on the basis of the results of the above analyses and previously reported data. The effects of various concentrations of cyclo(l-Pro–l-Leu) on the mortality rates of second-stage juveniles (J2) of M. incognita were investigated. Results showed that HPLC-purified cyclo(l-Pro–l-Leu) displayed nematicidal activities. The mortality rate of M. incognita J2 reached 84.3% after 72 h of exposure to 67.5 mg/L cyclo(l-Pro–l-Leu). The lowest egg-hatching rate (9.74%) was observed after 8 days of incubation with 2000 mg/L cyclo(l-Pro–l-Leu). An egg-hatching rate of 53.11% was obtained under the control treatment (sterile distilled water). However, cyclo(l-Pro–l-Leu) did not elicit chemotaxis activity to M. incognita. This is the first work to investigate the anti-M. incognita characteristics of cyclo(l-Pro–l-Leu).