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This study was conducted to isolate and identify thermophilic and thermotolerant bacteria from Jordanian hot springs and to determine hydrolytic, antimicrobial, and anticancer activities of the isolates. Thirty bacterial isolates were recovered from water samples of five main local hot springs. Nineteen of the isolated colonies were light yellow and circular to rhizoid on nutrient agar; cells were Gram-positive, endospore-forming, and rod-shaped. Eleven isolates were Gram-negative non-spore forming rods. It was found that 21 isolates met the criteria of moderate thermophiles; all isolates were grown aerobically (JA5 was facultative anaerobes) at 40-60 °C, pH 6-9, and 0-4% salt concentration and most of these isolates were reacted positively with catalase and oxidase. The remaining nine isolates were thermotolerant. Depending on the 16S rRNA gene sequences of the isolates, it was found that 19 thermophilic isolates have 97-100% sequence homology to the genus Bacillus; eight isolates were closely related to the thermophilic genus Geobacillus showing 97-100% homology to G. stearothermophilus ATCC 7953. The isolate JM2 shares 99% sequence homology with Thermomonas hydrothermalis. Remarkably, it was found that the 16S rDNA sequence of isolate JZ9 were highly similar (99% identity) to the thermophilic bacterium Caldimonas hydrothermale. To our knowledge, this is the first record of Caldimonas isolation from Jordanian hot springs. A wide spectrum of hydrolytic activities for protease, lipase, xylanase, cellulase, amylase, and pectinase was detected from the obtained isolates. It was found that JM1, JS3, and JZ11 isolates produced all tested enzymatic activities. Antimicrobial activities were only exhibited by three isolates (thermophilic JH1 and JM11 and thermotolerant JS3). Results indicated that three thermophilic Bacillus isolates (JA2, JM11, and JM12) produced selective cytotoxicity against human leukemia cell line K562. Therefore, many of the obtained isolates in this study can be considered as a promising source of effective agents that may be used for medical, pharmaceutical, and industrial purposes. [ABSTRACT FROM AUTHOR]