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With the planet rapidly urbanizing, understanding the ecological effects of urbanization is a grand challenge for modern biology. For example, increased city temperatures known as the urban heat island effect, disproportionately impact nocturnal taxa and this consideration is widely overlooked. Slight shifts in the thermal microclimate have a cascade of ramifications that directly impact species density and distribution. Animal behavior is a trait that may explain why some species thrive after urbanization when others go locally extinct. In this study we followed 22 adult females of the western black widow, Latrodectus hesperus, from both urban and undisturbed Sonoran Desert habitats. We began looking for differences between urban and desert spiders under field conditions: boldness, voracity, web size and body condition. Both urban and desert spiders were then brought to the laboratory to see how their behavior changed. We found no behavioral differences between urban and desert spiders in the field or the laboratory. We did find that spider behavior differed between the field and the laboratory. Specifically, boldness in the laboratory was significantly lower compared to the field. Voracity was more repeatable in the laboratory versus the field, and boldness was strongly positively correlated with voracity in the laboratory, but not in the field. These behavioral shifts from the field to the laboratory favor the conclusion that black widow behavior is highly plastic and context dependent. Lastly, we monitored web temperature of black widow microhabitat continuously for an entire year using thermochron data loggers. We found microhabitat temperatures differences between urban and desert sites were greatest at night and absent during the daytime. We uncovered a seasonal effect with the highest magnitude temperature difference occurring during the springtime. Additionally, behavior was significantly correlated with field temperatures; the boldest spiders come from the warmest webs. However, we found little evidence that temperature predicts spider body condition or voracity, and body condition does not predict its behavioral expression. Our results highlight the importance of studying animal behavior to increase understanding of the factors that shape distribution and density in a lethal pest species.