Spinach anthracnose caused by Colletotrichum spinaciae is a newly emergent disease that causes serious losses in the spinach fields of the Mediterranean Region of Turkey. The most popular spinach cv. Matador is very susceptible to the anthracnose. Currently, anthracnose disease is not effectively controlled in Turkey due to the lack of understanding of its epidemiology. In order to understand the effect of environmental parameters on anthracnose, the influences of inoculum concentration, temperature, leaf wetness duration, plant and leaf age on lesion development of spinach anthracnose was studied under controlled conditions. Seedlings of spinach cv. Matador at 4-leaf stage were inoculated with four inoculum concentrations, 102, 104, 106, 2 × 106 conidia/ml, and subjected to seven temperatures of 15, 18, 22, 25, 28, 30, and 33 °C, and nine leaf wetness durations of 3, 5, 10, 20, 24, 30, 44, 48, and 53 h. The mean number of lesions per plant increased at the rate of 50.7% with increasing inoculum density from 104 to 106 conidia per millilitre. Inoculum concentration with 106 conidia per ml of C. spinaciae was appropriate for infection and lesion development. Disease was observed at all temperatures and increased with wetness duration. The development of lesions per plant was observed more gradually when incubation temperature increased from 15 °C to 22 °C. The mean lesion number of anthracnose increased on spinach seedlings as wetness duration increased to 24 h and temperatures to 22 °C and 25 °C. Regression analysis revealed that there was a linear relationship between the increase in number of lesions per plant and wetness duration at 18, 25, and 30 °C. Optimum disease development was recorded at 22 °C with 24 h leaf wetness period. The highest number of lesions was observed at 45 d-old plants. In the same time, older leaves were more severely infected than younger leaves. This kind of information can be used to target key stages of an epidemic and may allow tailoring of spray programs or disease forecasts to cultivar with different levels of anthracnose resistance.