The simultaneous ultrasonic/microwave-assisted extraction (UMAE) method is potentially useful for the extraction of polysaccharides from Taxus chinensis var. mairei fruits (TCFPs). In this study, we used a response surface methodology to identify optimal TCFPs extraction conditions. Optimal parameters were determined as follows: a liquid to raw material ratio of 33 mL/g, an extraction time of 10 min, a microwave power level of 560 W, and a fixed ultrasonic power of 50 W. Under the optimized conditions, TCFPs yields obtained by UMAE were 4.33 ± 0.15%, a 1.79-fold increase compared with conventional heating reflux extraction (HRE). In addition, the extraction time used in UMAE was shorter than that required for HRE: 10 versus 90 min. UMAE is therefore a rapid and efficient method for the extraction of TCFPs. The inhibitory effect of TCFPs on S180 tumor growth in vivo was also studied. The tumor inhibition rate of TCFPs was 76.33%, indicating a tumor-inhibiting effect. Analysis of organ weights demonstrated that TCFPs exhibited no toxicity to liver, kidney, spleen, heart, or lung relative to a positive control group. TCFPs thus show antitumor activity with no organ toxicity.